With recent technical advances, multiple important signaling pathways that may be the causes of human malignancy have continuously been discovered and dissected. The vast majority of these signaling pathways involve reversible protein phosphorylation, and the information on the location and dynamics of phosphorylation provides important mechanisms on how the signaling networks function and interact. While translational research gradually shifts from lab models to clinical samples, with the ultimate goal of identifying cancer biomarkers, a simple and reliable phosphorylation assay method is still missing for routine detection of phosphorylation in complex and typically heterogeneous clinical samples. Through this NIH SBIR Phase II study we will further develop a novel strategy for phosphorylation assay, termed pIMAGO (phospho-imaging), which has recently been introduced and commercialized by us, into highly effective products for simple and routine phosphorylation assays. This novel design takes advantage of not only the quantum size properties of the soluble nanoparticles, but also of the multi-functionalized nature of the molecule, allowing for highly selective, sensitive and simple qualitative and quantitative assessment of protein phosphorylation without the use of either radioactive isotopes or expensive phosphospecific antibodies. Due to its size and unique properties, it also offers the capability for multiplexed detection of phosphorylation and total protein amount simultaneously. We propose to optimize the technology for universal on-membrane phosphoprotein detection in routine biological and biomedical research. In addition, we will develop a set of novel pIMAGO-based strategies for multiplexed detection of phosphorylation in antibody microarray and functional reversed phase array formats. The new applications will add another dimension to traditional biomedical research and development.